Lipotoxic cardiomyopathy increases the risk of heart failure in obese patients by adversely altering heart structure and function via toxic lipid specie mediated cellular stress and cell death. Increased fatty acid uptake and esterification in cardiomyocytes increases toxic lipid intermediates. These cardiotoxic lipid species such as diacylglycerol have recently been shown to deacidify lysosomes in cardiomyocytes by activating protein kinase C βII mediated NADPH oxidase 2 generation of superoxide that inhibits proton pumps on lysosomal membranes by S-nitrosylation. Autophagy, a lysosome dependent cellular survival process, is impaired upon cardiomyocyte lipid-overload due to inhibition of pH-dependent proteolytic autophagosome degradation in the lysosome. Subsequent accumulation of autophagic vesicles heightens cardiomyocyte sensitization to additional stresses of ischemia-reperfusion or ER dysfunction, culminating in impaired cardiac metabolic flexibility leading to cell death. Low cardiomyocyte regenerative capacity calls for strategies to preserve cell number in states of increased stress, such as lipid-induced impairment of autophagy. Lysosome-targeted reacidifying devices can provide an effective means to restore autophagic flux. In this thesis, a therapeutic strategy utilizing poly(DL-lactide-co-glycolide) (PLGA) nanoparticle degradation to reacidify lysosomes and revert cardiotoxic lipid specie induced blockade in autophagic flux in cardiomyocytes is presented. Endocytosed PLGA acidic nanoparticles were designed to rapidly degrade and release acidic monomers in lysosomes to restore pH dependent phosphatase and cathepsin L activity in cardiomyocytes with acute lipotoxicity. Optimized pre-palmitate treatment periods demonstrated that PLGA nanoparticles with polyethylenimine cationic surface coatings provide an effective restoration of autophagic flux in the presence of lipid-overload modeled by acute palmitate treatment in cardiomyocytes.

Des nouveaux ligands multidentes associant la fonction iminophosphorane (P=N) et des donneurs de types thiolates ou phenolates ont été développés au cours de la thèse. La synthèse de ces ligands présentement plusieurs avantages comme l'utilisation des produits bon marche ou la facilité de monter en échelle. Tous les ligands montrent une grande capacité de se coordiner avec plusieurs métaux, y comprise les métaux de transition, aluminium (group principal) et yttrium (terre-rare). Les complexes résultant de ces coordinations ont été étudiés théoriquement par les calculs DFT et expérimentalement par plusieurs techniques de spectroscopies électromagnétiques (RMN multi-noyaux, diffraction aux rayons X, RPE, UV-Vis, IR). Leur activités catalytiques ont été évaluées en tant que catalyseurs dans l'oligomerisation d'éthylène et la polymérisation de lactide pour des polymères biodégradables. En particulier, la synthèse des plusieurs ligands tétradentes phosphasalens a été mise au point. Ces ligands sont des homologues phosphorés des ligands Salen, qui, eux, ont fait l'objet d'études extensives ces dernières années. Les ligands phosphasalen ont été démontrés d'avoir une grande flexibilité et d'être excellent donneurs d'électrons. Grâce à ces propriétés, ils seront capables de stabiliser des métaux de haut degré d'oxydation. Un premier exemple très encourageant a été fait avec l'étude sur le complexe [Ni(III)-phosphasalen]. Ces ligands phosphasalens s'avèrent également très intéressants en catalyse. En effet, les complexes yttrium de ces ligands sont parmi les meilleurs initiateurs pour la polymérisation de lactide par ouverture de cycles, en terme de performance et stereoselectivity, alors que les complexes salen sont pas ou très peu active pour la même réaction. Ces études pionnières ouvrent plusieurs perspectives pour l'application des ligands phosphasalen en chimie de coordination et catalyse.

Construction of nanoassemblies from degradable components is desired for packaging and controlled release of active therapeutics, and eventual biodegradability in vivo. In this study, shell crosslinked micelles composed of biodegradable poly(lactide) (PLA) core were prepared by the self-assembly of an amphiphilic diblock copolymer synthesized by a combination of ring opening polymerization (ROP) and reversible addition-fragmentation chain transfer (RAFT) polymerization. Enzymatic degradation of the PLA cores of the nanoparticles was achieved upon the addition of proteinase K (PK). Kinetic analyses and comparison of the properties of the nanomaterials as a function of degradation extent will be discussed. Building upon our findings from selective-excavation of the PLA core, enzyme- and redox-responsive nanoparticles were constructed for the encapsulation and stimuli-responsive release of an antitumor drug. This potent chemotherapeutic, otherwise poorly soluble in water was dispersed into aqueous solution by the supramolecular co-assembly with an amphiphilic block copolymer, and the release from within the core of these nanoparticles were gated by crosslinking the hydrophilic shell region with a reduction-responsive crosslinker. Enzyme- and reduction-triggered release behavior of the antitumor drug was demonstrated along with their remarkably high in vitro efficacy. As cationic nanoparticles are a promising class of transfection agents for nucleic acid delivery, in the next part of the study, synthetic methodologies were developed for the conversion of the negatively-charged shell of the enzymatically-degradable shell crosslinked micelles to positively-charged cationic nanoparticles for the complexation of nucleic acids. These degradable cationic nanoparticles were found to efficiently deliver and transfect plasmid DNA in vitro. The hydrolysis of the PLA core and crosslinkers of the nanocarriers may provide a mechanism for their programmed disassembly within endosomes, which would in-turn promote endosomal disruption by osmotic swelling, and release of active therapeutics from the polymeric assemblies. In the last part, a comparative degradation study was performed between the anionic and cationic micellar assemblies in the presence of two model enzymes, and electrostatic interaction-mediated preferential hydrolysis was demonstrated between the oppositely-charged enzyme-micelle pairs. These findings may be of potential significance toward the design of charge-mediated enzyme-responsive nanomaterials that are capable of undergoing environmentally-triggered therapeutic release, disassembly or morphological alterations under selective enzyme conditions.

Poly(lactic acid), PLA, is a viable replacement to petroleum derived polymers due to its renewable feedstock, biodegradability and bioassimilability, yet improvements in its physical, thermal and mechanical properties are required before it can fully enter all commodity markets. This thesis investigates olefin cross-metathesis (CM) as a synthetic strategy to modify the properties of PLA. The use of novel lanthanide and actinide catalysts on the microstructure control of PLA are also explored. The Tebbe reagent was used in a new synthetic strategy to produce a novel olefin derivative of lactide (MML). Olefin CM of MML with hex-1-ene was successful but polymerisation pre- and post-CM was unsuccessful due to monomer instability. CM of another olefin derivative of lactide, 3-methylenated lactide (3-ML) was successful with aliphatic alkenes; hex-1-ene to dodec-1-ene. To overcome competing alcoholysis of the functionalised monomers, which prevented polymerisation, hydrogenation was used to remove the olefin entity followed by successful ring-opening polymerisation (ROP) to produce polymers of low glass-transition temperatures (Tg). Post-polymerisation CM on an olefin containing polymer P(β-heptenolactone) P(β-HL), with methyl acrylate and an epoxide, generated functionalised homopolymers with increased Tg’s. Co-polymerisation of lactide with β-HL generated novel gradient-copolymers. Olefin CM with 15 different cross-partners produced functionalised copolymers with different thermal properties. Based on this route a new methodology was created to introduce two unique functionalities into the polymer backbone by manipulation of the olefin reactivities. Finally, in a collaborative project, uranium and cerium catalysts, Me3SiOU(OArP)3 and Me3SiOCe(OArP)3 - designed out-with the group- were tested and compared as ROP catalysts for lactide. Both catalysts were active in living polymerisations of L-lactide and under immortal conditions the activity and rates of the catalysts were switched, accounted for by a change in the coordination sphere due to ligand displacement. ROP of rac-lactide using the uranium analogue produced heterotactic-biased PLA with a Pr = 0.79.

Este trabalho apresenta estudos sobre a síntese química do PEDOT com o objetivo de desenvolver diferentes arquiteturas e propriedades para melhorar a taxa de degradabilidade deste polímero. As estratégias foram as preparações de uma blenda, um copolímero e um nanocompósito. O estudo foi iniciado pela síntese química oxidativa do PEDOT (poli (3,4- etilenodioxitiofeno)) em microestruturas utilizando condições brandas e ambientalmente amigáveis, porém o material obtido não apresentou solubilidade e boas condições de se produzir um filme. Então foi sintetizado quimicamente o PEDOT dopado com PSS (poli estireno sulfonado) (PEDOT:PSS), o mesmo foi usado para preparar blendas com o PLGA (poli (ácido láctico-co-glicólico), para melhorar sua degradabilidade. Foi possível produzir um filme fino e nanoestruturado através de deposição eletrostática camada por camada (LBL) que pode ser utilizado para modificação de eletrodos ou de suportes tridimensionais para engenharia celular. Para garantir a degradabilidade do material, foi realizada a síntese de copolímeros de PEDOT e PLLA (poli(lactídeo)) em que foi variada a proporção de PEDOT na cadeia polimérica. Os copolímeros foram caracterizados por IV, RMN, UV, análises térmicas e submetidos a testes de degradabilidade e de viabilidade celular, apresentando excelentes resultados. Foi possível a obtenção de microfibras deste material. A outra alternativa estudada foi a síntese de um nanocompósito, preparado através da síntese química do PEDOT, partindo do monômero EDOT (3,4- etilenodioxitiofeno)usando HAuCl4 como oxidante e NaPSS como dopante e dispersante. O nanocompósito obtido foi caracterizado apresentando diâmetros médio próximos de 4 nm e com uma estrutura caroço-casca, apresentando nanopartícula de ouro como caroço e o polímero PEDOT:PSS como casca. Foram obtidos filmes deste material por deposição por evaporação de solvente, LBL, utilizando como policátion o PDDA (cloreto de poli (dialil dimetil amônio)) e quitosana, e por deposição eletroforética, que apresentou excelentes propriedades eletrocrômicas como rápidos tempos de respostas com bons contrastes ópticos / This work presents studies about the chemical synthesis of PEDOT (Poly(3,4-ethylenedioxythiophene)) with the aim of preparing different architectures and properties to improvement the degradability rate of this polymer. The strategies used to achieve this pupose were the preparation of polymer blends, copolymers and nanocomposites. The study was started by the chemical synthesis of microstructures PEDOT in mild and environment friendly conditions, but the material did not show solubility which enable film formation. Then, PEDOT was synthetized by chemical synthesis doped with PSS (poly(styrene sulfonic acid)), (PEDOT:PSS) to prepare blends with PLGA (poly(lactic-co-glycolic acid)) to improve its degradability. It was possible to prepare a thin and nanostructured film, by electrostatic layer-by-layer deposition (LBL), which could be used for electrodes or scaffold surface modification. In order to ensure the material\'s degradability, PEDOT and PLLA (poly(lactide)) copolymers were prepared, changing PEDOT proportion in the polymeric structure. The copolymers were characterized by, IR, NMR, UV, thermal analysis and then degradability and cell viability tests, which shown important results. Fibers were able to be obtained with these materials. The next strategy was the preparation of a nanocomposite by one-spot chemical synthesis, initiated by the monomer EDOT (3,4- ethylenedioxythiophene) using HAuCl4 as oxidant and NaPSS as both dopant and dispersant. The nanocomposite obtained was characterized showing diameter of around 4nm and a core-shell structure, with gold nanoparticle as core and PEDOT:PSS as the shell. Films were obtained by this material by casting, by LBL, using PDDA (Poly(diallyldimethylammonium) chloride) and chitosan as polycations, and by electrophoretic deposition. The latter method shows excellent characteristics as fast response time with a good optical contrast

Orientadores: Cecília Amélia de Carvalho Zavaglia, Marcos Akira d'Ávila / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Mecânica / Made available in DSpace on 2018-08-19T00:23:40Z (GMT). No. of bitstreams: 1 RodriguezPerea_GeraldineNancy_M.pdf: 1196408 bytes, checksum: 1a7f7c5e1320ddfd713867dbc7a1d5b6 (MD5) Previous issue date: 2011 / Resumo: Este trabalho consiste na obtenção pelo método de eletrofiação, de microfibras poliméricas e microfibras reforçadas com nanopartículas de hidroxiapatita. Este método foi utilizado, pois propicia a produção de membranas microporosas que possuem um grande potencial de aplicação na área de engenharia tecidual, especificamente em aplicações para regeneração óssea. Este trabalho teve como objetivo principal produzir fibras poliméricas com a intenção de comparar suas características com o nanocompósito de fibras poliméricas e nanopartículas de hidroxiapatita como reforço. Trabalhou-se com o poli(L-ácido lático) (PLLA) e nanopartículas de hidroxiapatita (HA) produzidas pelo processo sol-gel. As fibras e os nanocompósitos foram caracterizados pelos seguintes métodos: microscopia eletrônica de varredura (MEV), análise termogravimétrica (TGA), calorimetria exploratória diferencial (DSC) e espectroscopia na região do infravermelho por transformada de Fourier (FTIR). As fibras obtidas apresentaram diâmetros na faixa de 1 a 10 micrômetros. O objetivo de produzir membranas a partir de soluções de PLLA e nanocompósito PLLA/HA por eletrofiação foi atingido / Abstract: This work consists in obtaining polymeric microfibers and microfibers reinforced with nanoparticles of hydroxyapatite by the method of electrospinning. This method was used because it allows the production of microporous membranes that have great potential like application in tissue engineering, specifically in applications for bone regeneration. This work aimed to produce polymer fibers with the intention to compare their characteristics with the nanocomposite fibers with hydroxyapatite nanoparticles as reinforcement. The polymer used was poly (L-lactic acid) (PLLA) and nanoparticles of hydroxyapatite (HA) produced by the sol-gel process. The fibers and nanocomposites were characterized by the following methods: scanning electron microscopy (SEM), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), spectroscopy in the region of Fourier transform infrared (FTIR). The fibers obtained presented diameters in the range 1 to 10 micrometers. The goal of producing membranes from solutions of PLLA and nanocomposite PLLA / HA by electrospinning was reached / Mestrado / Materiais e Processos de Fabricação / Mestre em Engenharia Mecânica

The aim of this project was to create a novel biodegradable, synthetic scaffold that will provide the correct topographical cues for myoblast alignment and efficient differentiation into myotubes. Skeletal muscle repair after major surgery or serious burns is often overlooked leading to poor healing and consequent loss of power in movements of affected limbs. In order to overcome this problem a tissue engineered construct could be utilised as a grafting patch to encourage further regeneration and enhance possible power to the limb. Using a biodegradable polymer can provide structural support until the tissue is established, and will be excreted by the body's natural processes as it degrades. A synthetic polymer is desirable as it can reduce the risk of immunogenic responses thus reduce risk of graft rejection. For successful in vitro growth of skeletal muscle, the cells must be encouraged to arrange themselves into parallel arrays in order for efficient fusion and consequent contraction. By incorporating the correct topographical cues into the scaffold to promote contact guidance for cellular alignment this can be achieved. Electrospinning is a reliable technique which yields highly reproducible aligned fibres from the micro- to the nanoscale. This project focuses upon creating and characterising the electrospun scaffold, checking biocompatibility with myoblasts by monitoring the topography, residual solvent within the scaffold, the mechanical properties of the scaffold, and a brief investigation into the degradation profile of the electrospun fibres. The immunogenicity of the scaffold was investigated by monitoring cytokine release from macrophages. Myoblast morphology was monitored, as was the efficiency of the cells to differentiate and their potential to become contractile myofibres. Cellular adhesion to the scaffold was also looked into by measuring the expression of integrins during early and late adhesion and on substrates with different topographies. It was found that the electrospun scaffold did not contain a significant amount of residual solvent, and macrophages were not activated any more than on tissue culture plastic. Myoblasts responded to the topography of the aligned fibres by aligning along the length of the fibres, showing elongation and bi-axial cytoskeletal arrangement after just 30 minutes culture on the aligned fibres. This elongation prompted fusion and differentiation of the myoblasts to occur faster than cells which were not exposed to the aligned topography, and this global alignment was maintained in long term culture.

New methods for the preparation of porous biomedical scaffolds have been explored for applications in tissue engineering and drug delivery. Scaffolds with controlled pore morphologies have been generated which incorporate cyclodextrin-drug inclusion complexes as the drug delivery component. Supercritical CO2 was explored as the main processing fluid in the complex formation and in the foaming of the polymer scaffold. The co-solvents, ethanol, ethyl acetate and acetone, were explored in each stage, as needed, to improve the solvent power of CO2. The first goal was to promote cyclodextrin-drug complex formation. Complex formation by traditional methods was compared with complex formation driven by processing in supercritical CO2. Complex formation was promoted by melting the drug in supercritical CO2 or in CO2 + co-solvent mixtures while in the presence of cyclodextrin. Some drugs, such as piroxicam, are prone to degradation near the drug's ambient melting temperature. However, this approach using CO2 was found to circumvent drug thermal degradation, since drug melting temperatures were depressed in the presence of CO2. The second goal was to produce porous polymeric matrices to serve as tissue engineering scaffolds. Poly(lactide-<i>co</i>-glycolide) and poly(ε-caprolactone) were investigated for foaming, since these biomedical polymers are already commonly used and FDA approved. Polymer foaming with CO2 is an alternative approach to conventional solvent-intensive methods for porosity generation. However, two major limitations of polymer foaming using CO2 as the only processing fluid have been reported, including the formation of a non-porous outer skin upon depressurization and limited pore interconnectivity. Approaches to circumvent these limitations include the use of a co-solvent and controlling depressurization rates. The effect of processing parameters, including foaming temperatures and depressurization rate, as well as co-solvent addition, were examined in polymer foaming using CO2. Drug release dynamics were compared for foams incorporated with either pure drug, cyclodextrin-drug physical mixture or cyclodextrin-drug complex. Pore morphology, polymer choice and drug release compound choice were found to alter drug release profiles. / Ph. D.

Syntéza polylaktidu (PLA) polymerací za otevření kruhu cyklického monomeru (ROP) může být uskutečněna různými způsoby. Literatura uvádí více než 100 katalytických systémů, jejichž pomocí lze polylaktid a jiné biodegradabilní alifatické polyestery získat. Například organokovové katalyzátory na bázi Sn, Zn, Al atd. se po splnění své polymerační funkce stávají kontaminanty a pro humánní implantáty je použití takového materiálu diskutabilní. V současné době jsou v centru výzkumné pozornosti nové N-heterocyklické karbenové katalyzátory. Tyto „metal-free“ katalytické struktury jsou schopné reprodukovatelně řídit syntézu polymerů předem definované molekulové hmotnosti s definovanými koncovými skupinami a nízkou polydisperzitou, která je charakteristická pro živý průběh polymerace. Nabízí se možnost syntézy blokových kopolymerů a různorodých makromolekulárních architektur. Předložená diplomová práce se zabývá studiem polymerace cyklického monomeru D,L-laktidu katalyzované N-heterocyklickým karbenem. Polymerace byly vedeny v přítomnosti benzylalkoholu jako iniciátoru v roztoku THF. Byl sledován vliv složení reakčního systému monomer – iniciátor – katalyzátor. Dále byly připraveny polymery opticky čistého L-laktidu s makroiniciátory PEG s Mn = 1000 a 2000 g/mol. Střední číselná molekulová hmotnost (Mn) a polydisperzita (PDI) byly stanoveny pomocí GPC. Definovatelnost koncových skupin vybraných polymerů byla prokázána pomocí 1H NMR.

This master thesis deals with the synthesis of lactide from lactic acid esters. The theoretical part focuses on the current understanding of technology of lactic acid, its alkyl esters and the cyclic lactic acid dimer - lactide, which is mainly used as a precursor for the synthesis of high moleculat weight biodegradable polylactide (PLA) and other copolymers. Experimental part is then focused on the synthesis of lactide from lactic acid alkyl esters, especially ethyl lactate. Examines the design of a suitable apparatus for this synthesis and the application of the most appropriate reaction conditions (temperature, pressure, catalysis) to optimize the preparation of the desired product. In the end, parameters of prepared lactide are characterized by using of various analytical techniques.